Nowadays, there is a high demand for sensitive and selective real-time analytical methods suitable for a wide range of applications, from personalized telemedicine, drug discovery, food safety, and quality control, to defense, security, as well as environmental monitoring. Biosensors are analytical devices able to detect bio-chemical analytes (e.g., neurotransmitters, cancer biomarkers, bio-molecules, and ions), through the combination of a bio-recognition element and a bio-transduction device. The use of customized bio-recognition elements such as enzymes, antibodies, aptamers, and ion-selective membranes facilitates achieving high selectivity. Among the different bio-transduction devices currently available, electrolyte-gated field-effect transistors, in which the dielectric is represented by an ionic liquid buffer solution containing the targeted analyte, are gaining increasing attention. Indeed, these biotransduction devices are characterized by superior electronic properties and intrinsic signal amplification that allow the detection of a wide range of biomolecules with high sensitivity (down to pM concentration). A promising semiconducting material for bio-transduction devices is represented by carbon nanotubes, due to their unique electrical properties, nanosize, bio-compatibility, and their simple low-cost processability. This work provides a comprehensive and critical review of electrolyte-gated carbon nanotube field-effect transistor-based biosensors. First, an introduction to these bio-sensing devices is given. Next, the device configurations and operating principles are presented, and the most used materials and processes are reviewed with a particular focus on carbon nanotubes as the active material. Subsequently, different functionalization strategies reported in the literature, based on enzymes, antibodies, aptamers, and ion-selective membranes, are analyzed critically. Finally, present issues and challenges faced in the area are investigated, the conclusions are drawn, and a perspective outlook over the field of bio-sensing technologies, in general, is provided.